Theophylline is a drug frequently administered for treatment of asthma and pulmonary diseases. For the drug to be used successfully without serious side-effects, it must be frequently and carefully monitored in a patient because it has a relatively narrow therapeutic range of use, that is, 1-2 mg/dl.
Numerous techniques have been used to determine the amount of theophylline in human serum. Most of these techniques have serious drawbacks. For example, known spectrophotometric methods require large sample volumes, extensive pretreatment and suffer from interferences by similarly structured xanthines, such as caffeine and theobromine. Known gas chromatographic methods are more specific, but require derivatization and are time consuming.
Nonisotropic immunoassay techniques are most frequently used because they provide rapid results and are simple to use. Although satisfactory sensitivity has been generally obtained with immunoassay techniques, it has been found recently that they may produce highly elevated results depending upon a patient's renal condition and the specificity of the antibody used in the assay. Moreover, immunoassays require the use of generally costly reagents which have limited stability.
High performance liquid chromatography techniques are also known. These techniques vary in specificity depending upon whether pretreatment of the test sample is carried out. Organic extraction steps are necessary to improve the accuracy and specificity of the assay. Many chromatography methods are susceptible to interferences from a number of substances including some common antibiotics. Other disadvantages include the need for expensive instrumentation and a specialized technical staff to perform the assays.
It is known that theophylline can be determined by measuring its inhibitory effect on alkaline phosphatase activity. However, when assaying human biological fluids in this manner, it is known that endogenous alkaline phosphatase can affect the assay and render inaccurate results on the low side. Endogenous alkaline phosphatase must then be destroyed or removed in some manner prior to the assay to avoid this problem.
In commonly assigned U.S. Ser. No. 900,069, now U.S. Pat. No. 4,782,017, filed Aug. 25, 1986 by Frickey et al entitled "Analytical Element and Method for Theophylline Determination Using Buffer in Spreading Zone", an element is described that has an isoenzyme for alkaline phosphatase and a buffer for maintaining the pH at 9 or less during the assay. Substantially all of the buffer is located in the spreading layer. No buffer is included in the subbing layer of the element.
While this element provides a simple and rapid assay for theophylline in which endogenous alkaline phosphatase exhibits a reduced effect, there is a need for further improvement. The element described above has a limited range and significant alkaline phosphatase bias.